Pharmaceutical combination comprising a cip2a silencing agent for use in the treatment of a hyperproliferative disorder, preferably one with impaired p53 function

ABSTRACT

The invention is based on a finding that silencing CIP2A (KI-AA1524) gene sensitizes cancer cells for apoptosis-inducing activity of certain small molecule chemotherapeutic agents. Thus, the invention is directed to a respective combination therapy, sensitization method and pharmaceutical compositions. The invention further relates to a method of selecting cancer therapy for a subject on the basis of CIP2A and p53 expression and/or protein activity in a sample obtained from said subject.

FIELD OF THE INVENTION

This invention relates to the field of combination cancer therapeutics.

BACKGROUND OF THE INVENTION

Cancer is a devastating disease afflicting all communities worldwide. Ithas been estimated that 1 out of 2 men and 1 out 3 women will developsome form cancer within their lifetime.

Interestingly, it has been recently established that, regardless of thephenotypic variability between different cancer types, perturbation oflimited number of genetic elements is sufficient to induce cellulartransformation in many different human cell types (reviewed in (Zhao etal., 2004)). Experimentally, it was demonstrated that activation of Rasand telomerase (TERT), along with inactivation of the tumor suppressorproteins p53 and Retinoblastoma protein (Rb) can immortalize a varietyof human cell types, which can subsequently transform to a tumorigenicstate in response to inhibition of protein phosphatase 2A (PP2A).Therefore, these common genetic elements could be considered as masterregulators of cancer development (Zhao et al., 2004).

PP2A is a widely conserved protein serine/threonine phosphatase (PSP)that functions as a trimeric protein complex consisting of a catalyticsubunit (PP2Ac or C), a scaffold subunit (PR65 or A), and one of thealternative regulatory B subunits. As described above, recentexperimental evidence has firmly established that inhibition of PP2Aactivity is a prerequisite for human cell transformation (reviewed in(Westermarck and Hahn, 2008)). Nevertheless, very little is known aboutmechanisms regulating PP2A complex composition and/or activity in vivo.Identification of PP2A inhibiting mechanisms might provide opportunitiesfor development of novel class of cancer therapeutics reactivating PP2Atumor suppressor activity. This idea would be similar to cancer therapyapproaches aiming at re-activation of tumor suppressor activity of p53by small-molecules such as Nutlin-3 (Vassilev et al., 2004).

In 2007 a novel PP2A inhibitor protein designated Cancerous inhibitor ofPP2A (CIP2A) was identified (Junttila et al., 2007). CIP2A interactswith PP2A and with one of the most important oncogenic transcriptionfactors MYC. Moreover, siRNA-mediated depletion of CIP2A markedlyincreased PP2A activity in the MYC-PP2A complex and resulted in MYCserine 62 dephosphorylation and MYC protein degradation. It has alsobeen demonstrated that CIP2A is required for the malignant cellulargrowth and for in vivo tumor formation (Junttila et al., 2007; Khanna etal., 2009; Westermarck and Hahn, 2008). Moreover, recent work hasdemonstrated overexpression of CIP2A in several common humanmalignancies and validated its role as a clinically relevant humanoncoprotein (Khanna et al., 2009; Westermarck and Hahn, 2008). Thus,these results demonstrate that CIP2A is a novel human oncoprotein thatinhibits PP2A in human malignancies.

Cell killing and/or apoptosis are the preferable endpoints for cancertherapy regimens. On the other hand, either intrinsic or acquiredresistance is the major problem related to currently usedchemotherapies. Thus, although at least some of the mechanismsunderlying malignancy have been revealed, there exists a need in the artfor the development of medicaments for hyperproliferative diseases andespecially cancer. Activation of tumor suppressor protein p53 mediatesapoptosis induction of cells in response to variety of thechemotherapeutics in clinical use. (Chari et al., 2009). However, as p53function is impaired in approximately 50-70% of human cancers this is animportant cause of chemotherapy resistance. (Chari et al., 2009). p53 isinactivated in cancer in most cases either by genetic mutations or byoverexpression of negative regulators of p53 such as MDM2 or viralproteins such as human papillomavirus (HPV) 16 E6 protein. Thus,approaches that sensitizes those cancer cells that harbour functionallyimpaired p53 to chemotherapy are clearly needed in order to overcome thedrug resistance (Chari et al., 2009).

BRIEF DESCRIPTION OF THE INVENTION

In one aspect, the invention provides a combination of at least one typeof a CIP2A silencing agent and a chemotherapeutic agent selected fromthe group consisting of small molecule tyrosine kinase inhibitors, PARPinhibitors, CHK1 inhibitors, glucosinolates, alkylating agents, plantalkaloids, PI3K/mTOR inhibitors, histone deacetylases, DNA-PKinhibitors, STAT inhibitors, antimetabolites, and surviving inhibitorsfor use as a medicament in the treatment of hyperproliferative disorderscomprising cells with impaired p53 function. Suitable agents of eachcategory agents are set forth in claim 1.

In a further aspect, the invention provides pharmaceutical compositioncomprising a combination of CIP2A silencing agent and a chemotherapeuticagent according to the present invention and at least onepharmaceutically acceptable carrier.

In a still further aspect, the present invention provides a method ofsensitizing hyperproliferative cells to a chemotherapeutic agent bysilencing CIP2A gene in a human or animal subject in need of suchsensitization.

In an even still further aspect, the invention provides a method oftreating a hyperproliferative disease in a human or animal subject inneed of such treatment by administering at least one type of CIP2Asilencing agent and a compound selected from the group consisting ofsmall molecule tyrosine kinase inhibitors, PARP inhibitors, CHK1inhibitors, glucosinolates, alkylating agents, plant alkaloids,PI3K/mTOR inhibitors, histone deacetylases, DNA-PK inhibitors, STATinhibitors, antimetabolites, and surviving inhibitors concomitantly,simultaneously, or subsequently to said subject. Suitable compounds ofeach category are set forth in claim 8.

Furthermore, one aspect of the present invention provides a method ofselecting a cancer therapy for a subject in need of such therapy,wherein the method comprises evaluating CIP2A and p53 expression and/orprotein activity in a sample obtained from said subject, and selectingmonotherapy by at least one chemotherapeutic agent for subjects whosesample is negative for CIP2A expression and/or activity and impaired forp53 activity, and selecting a combination therapy according to thepresent embodiments for subjects whose sample is positive for CIP2Aexpression and impaired for p53 activity.

In some embodiments of the above aspects, said CIP2A silencing agent isselected from the group consisting of an sRNA molecule, DsiRNA molecule,artificial miRNA precursor, shRNA molecule, antisense oligonucleotide,ribozyme, and agent preventing CIP2A function towards PP2Ac. In furtherembodiments, the CIP2A silencing agent comprises as a nucleic acidsequence selected from the group consisting of SEQ ID NO:s 1 to 25, andsequences having at least 80% sequence identity to said SEQ ID NO:s 1 to25 and retaining their CIP2A silencing activity.

In some embodiments, of the above aspects, the hyperproliferativedisorder to be treated is selected from a group consisting of psoriasis,myocardial hypertrophy, benign tumors, solid cancers and haematologicalcancers. Non-examples of said solid cancers include squamous cellcarcinomas of the head and neck, colon cancer, gastric cancer, breastcancer, ovarian cancer, prostate cancer, cervical cancer, esophagealcancer, lung cancer, liver cancer, brain cancer, glioma, astrocytoma,and glioblastoma, whereas non-limiting examples of haematologicalcancers include acute and chronic T-cell and B-cell leukemias andlymphomas.

Other specific embodiments, objects, details, and advantages of theinvention are set forth in the dependent claims, following drawings,detailed description and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following the invention will be described in greater detail bymeans of preferred embodiments with reference to the attached drawings,in which

FIG. 1 shows the level of apoptosis (i.e. Caspase 3/7 activity), inducedin the human derived glioma cell line, T98G, following the inhibition ofCIP2A with siRNA in combination with standard chemotherapeutic agentswhen compared to SCR siRNA transfected cells alone.

FIG. 2 shows the efficacy of CIP2A siRNA used in combination withvarious chemotherapy drugs to reduce cell viability as determined usingthe CTG assay, in the human derived cervical cancer cell line, HeLa,when compared to SCR siRNA alone.

FIG. 3 demonstrates the efficacy of various chemotherapeutic drugs toinduce apoptosis in primary mouse lymphoma cells derived from CIP2Adeficient (CIP2A^(−/−)) mice or CIP2A expressing mice (CIP2A^(+/+))mice, as determined using the Caspase 3/7 assay.

FIG. 4 demonstrates that cell viability, determined using the CTG assay,is significantly reduced in primary lymphoma cells derived from CIP2Adeficient (CIP2A^(−/−)) mice when compared to cells expressing CIP2A,following the treatment of cells with various chemotherapy drugs.

FIG. 5 is a Caspase 3/7 assay which measures the level of apoptosis(i.e. Caspase 3/7 activity) induced in the human derived gastric cancercell line, MKN28, following the inhibition of CIP2A with siRNA incombination with standard chemotherapeutic agents when compared to SCRsiRNA transfected cells alone.

FIG. 6 demonstrates that apoptosis (i.e. Caspase 3/7 activity) is notinduced in the human derived breast cancer cell line, MCF-7, followingthe inhibition of CIP2A with siRNA in combination with standardchemotherapeutic agents when compared to SCR siRNA transfected cellsalone.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on a surprising finding that silencingCIP2A gene sensitizes cancer cells compromised for p53 tumor suppressorprotein function for apoptosis-inducing activity of certain smallmolecule chemotherapeutic agents. Concomitant silencing of CIP2A geneand administration of said chemotherapeutic agent results in eitheradditive or synergistic increase in the level of apoptosis and/or othertype of cell death in cells in which p53 function has become inhibited.On the other hand, the invention shows that CIP2A negative lymphomacells expressing mutated p53 are more sensitive to monotherapy with saidchemotherapeutic agents. Thus, in one aspect, the invention provides acombination therapy of CIP2A depletion and said chemotherapeutic agents,while in another aspect the invention provides a method forstratification of cancer patients for chemotherapy by detection of CIP2Aand p53 status of patient samples.

The present patient stratification method comprises determining theCIP2A and p53 status of a cancer tissue sample obtained from saidpatient. Patients with CIP2A negative and p53 inactivated cancer cellsshould be selected for monotherapy treatment with chemotherapeuticagents, whereas patients with CIP2A positive and p53 inactivated cancercells should be treated with the present combination therapy, i.e. CIP2Ainhibition together with administration of certain chemotherapeuticagents.

The level of CIP2A in a cancer tissue sample or a bodily fluid may bedetermined by various means. For instance, the level of the CIP2Aprotein in a tissue or body fluid may be quantified by i) determiningthe CIP2A mRNA expression from said tissue or body fluid by RT-PCR, orby a hybridizing technique, or ii) subjecting the tissue or body fluidexpected to contain the protein CIP2A to an antibody recognizing saidCIP2A, and detecting and/or quantifying said antibody, or subjectingsaid tissue or body fluid to analysis by proteomics techniques. Suitablehybridizing techniques include, for example DNA hybridization andnorthern blot. The detection or quantification of the antibody can beperformed according to standard immunoassay protocols, such aslabel-linked immunosorbent assays, western blot and immunohistochemicalmethods.

Impaired function of p53 in a cancer tissue or a bodily fluid sample maybe determined by standard methods known to those skilled in the art. Themethod of choice depends, at least partly, on the mechanism underlyingthe impaired p53 function. For instance, detection of p53 inactivatingmutations may be performed by hybridisation techniques or by DNA or RNAsequencing or by RT-PCR analysis of the RNA or DNA, as well known to aperson skilled in the art. Overexpression of negative regulators of p53such as MDM2 or viral proteins such as human papillomavirus (HPV) 16 E6protein, may be determined the same way as the level of CIP2A.

The patient stratification method can be carried out by determining thestatus of CIP2A and p53 alone or with the same in combination with otherproteins or genes.

CIP2A gene silencing may be obtained by any suitable method known in theart including, but not limited to, RNA interference (RNAi). The mostcommon approach for RNAi-based gene silencing is the use of smallinterfering RNA (siRNA).

The principle of siRNA is extensively presented in literature. Asexamples can be mentioned the US patent publications 2003/0143732,2003/0148507, 2003/0175950, 2003/0190635, 2004/0019001, 2005/0008617 and2005/0043266. A siRNA duplex molecule comprises an antisense region anda sense strand wherein said antisense strand comprises sequencecomplementary to a target region in an mRNA sequence encoding a certainprotein, and the sense strand comprises sequence complementary to thesaid antisense strand. Thus, the siRNA duplex molecule is assembled fromtwo nucleis acid fragments wherein one fragment comprises the antisensestrand and the second fragment comprises the sense strand of said siRNAmolecule. In other words, siRNAs are small double-stranded RNAs(dsRNAs). The sense strand and antisense strand can be covalentlyconnected via a linker molecule, which can be a polynucleotide linker ora non-nucleotide linker. The length of the antisense and sense strandsmay vary and is typically about 19 to 21 nucleotides each. In somecases, the siRNA may comprise 22, 23 or 24 nucleotides.

Another approach for RNAi-based CIP2A silencing is to use longer,typically 25-35 nt, Dicer substrate siRNAs (DsiRNAs), which in somecases have been reported to be more potent than correspondingconventional 21-mer siRNAs (Kim et al., 2005). DsiRNAs are processed invivo into active siRNAs by Dicer.

In a cell, an active siRNA antisense strand is formed and it recognizesa target region of the target mRNA. This in turn leads to cleaving ofthe target RNA by the RISC endonuclease complex (RISC=RNA-inducedsilencing complex) and also in the synthesis of additional RNA by RNAdependent RNA polymerase (RdRP), which can activate Dicer and result inadditional siRNA duplex molecules, thereby amplifying the response.

As used herein, the term “dsRNA” refers to both siRNAs and DsiRNAs.

Typically, but not necessarily, the antisense strand and the sensestrand of dsRNA both comprise a 3′-terminal overhang of a few, typically1 to 3 nucleotides. The 3′ overhang may include one or more modifiednucleotides, such as a 2′-O-methyl ribonucleotide. The 5′-terminal ofthe antisense is typically a phosphate group (P). The dsRNA duplexeshaving terminal phosphate groups (P) are easier to administrate into thecell than a single stranded antisense. In some cases, the 5′-terminal ofthe sense strand or of both antisense and sense strands may comprise a Pgroup.

Normal, unmodified RNA has low stability under physiological conditionsbecause of its degradation by ribonuclease enzymes present in the livingcell. If the oligonucleotide shall be administered exogenously, it ishighly desirable to modify the molecule according to known methods so asto enhance its stability against chemical and enzymatic degradation.

Modifications of nucleotides to be administered exogenously in vivo areextensively described in the art (e.g. in US 2005/0255487, incorporatedherein by reference). Principally, any part of the nucleotide, i.e theribose sugar, the base and/or internucleotidic phosphodiester strandscan be modified. For example, removal of the 2′-OH group from the riboseunit to give 2′-deoxyribosenucleotides results in improved stability.Prior disclosed are also other modifications at this group: thereplacement of the ribose 2′-OH group with alkyl, alkenyl, allyl,alkoxyalkyl, halo, amino, azido or sulfhydryl groups. Also othermodifications at the ribose unit can be performed: locked nucleic acids(LNA) containing methylene linkages between the 2′- and 4′-positions ofthe ribose can be employed to create higher intrinsic stability.

Furthermore, the internucleotidic phosphodiester linkage can, forexample, be modified so that one or more oxygen is replaced by sulfur,amino, alkyl or alkoxy groups. Also the base in the nucleotides can bemodified.

Preferably, the oligonucleotide comprises modifications of one or more2′-hydroxyl groups at ribose sugars, and/or modifications in one or moreinternucleotidic phosphodiester linkages, and/or one or more lockednucleic acid (LNA) modification between the 2′- and 4′-position of theribose sugars.

Particularly preferable modifications are, for example, replacement ofone or more of the 2′-OH groups by 2′-deoxy, 2′-O-methyl, 2′-halo, e.g.fluoro or 2′-methoxyethyl. Especially preferred are oligonucleotideswhere some of the internucleotide phoshodiester linkages also aremodified, e.g. replaced by phosphorothioate linkages.

In some embodiments, dsRNAs may contain one or more synthetic or naturalnucleotide analogs including, but not limited to, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, andpeptide-nucleic acids (PNAs) as long as dsRNAs retain their CIP2Asilencing ability.

It should be stressed that the modifications mentioned above are onlynon-limiting examples.

One of the challenges related to RNAi is the identification of a potentdsRNA for the corresponding mRNA. It should be noted that genes withincomplete complementarity are inadvertently downregulated by the dsRNA,leading to problems in data interpretation and potential toxicity. Thishowever can be partly addressed by carefully designing appropriatedsRNAs with design algorithms. These computer programs sieve out giventarget sequence with a set of rules to find sequence stretches with lowGC content, a lack of internal repeats, an NU rich 5-end and high localfree binding energy which are features that enhance the silencing effectof dsRNA.

In order to identify agents useful in the present invention, severaldifferent CIP2A siRNAs were designed by using commercial andnon-commercial algorithms. To this end, full length cDNA sequence ofCIP2A (KIAA1524) was loaded to siRNA algorithm programs (Eurofins MWGOperon's Online Design Tool) and stand-alone program developed by Cui etal. (Cui et al., 2004). Further, algorithm generated siRNA sequenceswere then screened trough genome wide DNA sequence alignment (BLAST) toeliminate siRNAs which are not free from off-targeting. In other words,all those siRNAs which had even short sequence regions matching withother genes than target gene (CIP2A) were considered invaluable forfurther use.

Obtained siRNAs were then transfected to different cell lines and theircapacity to degrade mRNA and further deplete translation of CIP2A wasstudied at protein level by measuring the amount of CIP2A protein aftersiRNA treatment with CIP2A specific antibodies (Table 1).

TABLE 1  CIP2A specific siRNAs % CIP2A inhibition SEQ IDsiRNA sense sequence (protein NO (5′ to 3′) level) 15′-AACATAAGTGCTTCACTGATCTT-3′ Moderate 2 5′-AACTGTGGTTGTGTTTGCACTTT-3′High 3 5′-GGUUGCAGAUUCUGAAUUATT-3′ Moderate 4 5-AAUGCCUUGUCUAGGAUUATT-3′Low 5 5′-ACCAUUGAUAUCCUUAGAATT-3′ High

Suitable dsRNAs include those having a greater than 80% sequenceidentity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequenceidentity with SEQ ID NO:s 1 to 5, as long as they have similar orenhanced binding properties and CIP2A silencing activity as thereference dsRNAs.

Still further CIP2A specific dsRNAs suitable for use in variousembodiments of the present invention can be designed and synthetizedaccording to methods known in the art. Any such isolated dsRNA must besufficiently complementary to CIP2A cDNA sequence in order to silenceCIP2A gene.

Artificial microRNA (miRNA) precursors are another class of small RNAssuitable for mediating RNAi. Typically, artificial miRNA precursors areabout 21-25 nucleotides in length, and they may have 1 to 3, typically2, overhanging 3′ nucleotides. CIP2A silencing artificial miRNAprecursors may be designed and synthetized by methods known in the art.

Short-hairpin RNAs (shRNAs) are still another way of silencing CIP2A.ShRNAs consist of i) a short nucleotide sequence, typically ranging from19 to 29 nucleotides, derived from the target gene; ii) a loop,typically ranging between 4 to 23 nucleotides; and iii) a shortnucleotide sequence reversely complementary to the initial targetsequence, typically ranging from 19 to 29 nucleotides. ShRNA expressioncassette may also contain sequences that increase the RNA interferenceactivity. Non-limiting examples of such sequences are microRNA sequenceof mir-30 as shown by Silva et al (Silva et al., 2005).

CIP2A silencing shRNAs may be designed and synthetized by means andmethods known to a skilled person. Non-limiting examples of suitablesense sequences (i.e. nucleotide sequences i) above) for use in CIP2AshRNAs are listed in Table 2. ShRNAs depicted in SEQ ID NO:6 to SEQ IDNO:9 are available e.g. from Origene, while shRNAs depicted in SEQ IDNO:10 to SEQ ID NO:25 are available e.g. from Open Biosystems.

TABLE 2  Sense sequences of CIP2A specific shRNAs SEQ IDsiRNA sense sequence NO (5′ to 3′) 6 5′-GATAGCAATGATCCACAGTTTAAGTGGTG-3′7 5′-CTTTGTCGGCACAATCTTTCTGTTCAAAC-3′ 85′-GTACTTGGAGAAAGTATAGCAGCAAACAA-3′ 9 5-CAGTTGACCTACTGATGGATCTCCTTAAG-3′10 5′-CGCAGATTCTGAATTATGCAAA-3′ 11 5′-AGCACATAAAGACATTGAGTAA-3′ 125′-ATTCCTGATAGATCACATTCAA-3′ 13 5′-CACGTCAGATAATAGAGAACAA-3′ 145′-CATGGATGTATATGAAATGAAA-3′ 15 5′-CCGGCACAATCTTTCTGTTCAA-3′ 165′-AGCACATAAAGACATTGAGTAA-3′ 17 5′-CGCAAACTTGCTGCTGATGTAA-3′ 185′-CCGGCACAATCTTTCTGTTCAA-3′ 19 5′-CGCAGCAAGTTGAATCAGAAA-3′ 205′-CCACAGTTTAAGTGGTGGAAA-3′ 21 5′-GCTAGTATGTTGAGAGAAGTT-3′ 225′-GCTAGTAGACAGAGAACATAA-3′ 23 5′-GACAGAAACTCACACGACTAT-3′ 245′-CCACAGTTTAAGTGGTGGAAA-3′ 25 5′-CGGCACAATCTTTCTGTTCAA -3′

Suitable shRNAs include those having a greater than 80% sequenceidentity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequenceidentity with SEQ ID NO:s 6 to 25, as long as they have similar orenhanced binding properties and CIP2A silencing activity as thereference shRNAs.

CIP2A silencing may also be obtained by antisense therapy, whererelatively short (typically 13-25 nucleotides) synthetic single-strandedDNA or RNA oligonucleotides inactivate CIP2A gene by binding to acorresponding mRNA. Antisense oligonucleotides may be unmodified orchemically modified. In some embodiments, the hydrogen at the2′-position of ribose is replaced by an O-alkyl group, such as methyl.In further embodiments, antisense oligonucleotides may contain one ormore synthetic or natural nucleotide analogs including, but not limitedto PNAs.

Furthermore, CIP2A silencing may obtained by ribozymes cleaving theCIP2A mRNA. The ribozyme technology is described, for example, by Li etal. (Li et al., 2007).

As used herein, the term “CIP2A silencing” refers to complete or partialreduction of CIP2A gene expression. In some embodiments, CIP2A geneexpression is reduced by at least 50%, or at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% when CIP2A-specific dsRNA, artificial miRNA precursor,shRNA, antisense oligonucleotide, ribozyme, or any combination thereofis introduced into a human or animal subject.

In some embodiments, CIP2A silencing may be obtained by blocking orinhibiting the interaction between CIP2A and PP2A, especially thecsubunit of PP2A, thus preventing CIP2A function towards PP2Ac at least50%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Such blocking orinhibiting agents include, but are not limited to, recombinantly orchemically produced modified or unmodified peptides and partialpeptides, as well as non-peptide molecules, such as small moleculechemical compounds. Methods for identifying such agents have beendisclosed e.g. in WO 2009/100173 and US 2009/239244.

Chemical compounds suitable for use in various embodiments of thepresent invention include those listed in Table 3 and any stereoisomers,salts, solvates, or prodrugs thereof.

TABLE 3 Chemical Compounds Chemotherapy Molecular Class of Drug DrugSynonyms Formula Small Molecute Tyrosine Lapatinb INN. Tycerb ®C₂₉H₂₈CFN₄O₄S Kinase Inhibitors Tandutinb C₃₁H₄₂N₆O₄ Vandatanb INN.ZD6474, Zactima C₂₂H₂₄BrFN₄O₂ Dasatinb BMS 354825 Sprycal C₂₂H₂₆ClN₂O₂SPKC-412 Midostauin C₃₅H₃₀N₄O₄ Benzoylstaurosporine H-7 C₁₄H₁₇N₃O₂S•2HClSunlinib SU11248 C₂₂H₂₇FN₄O₂ PARP Inhibitors ABT-888 VelparibC₁₃H₁₈N₄O•2HCl AG-014699 C₁₈H₁₆FN₂O•H₃PO₄ IND-71677 BSI-201, triparb,NSC- C₂H₃IN₂O₃ 746045 Olaparib C₂₅H₂₃FN₄O₃ PARP inhibitor III3,4-Dihydro-5[4-(1- C₁₈H₃₅N₂O₂ (DPQ) piperindinyl)butoxy]-1(2H)-isoquinoine CHK1 Inhibitors UCN-01 7-HydroxystaurosporineC₂₈H₂₈N₄O₄ AZD7762 5-(3-Fluorophenyl)-3- C₁₇H₂₀CFN₄O₂Sureidothiophene-N-[(S)- piperidin-3-yl]-2- carboxamide PF-477736PF-0044736 C₂₂H₂₅N₇O₂ SB 218078 C₂₄H₁₅N₃O₃ G86 976 PD 406976, Go 6 976C₂₄H₁₈N₄O Glucosinolates Indol-3-carbinol I3C C₉H₉NO Alkylating AgentsCisplatin CDDP, Plainol ® Cl₂H₈N₂Pl Plant Alkaloids Temozolomide TMZ,Temodal, C₅H₈N₈O₂ Temodar ® PI3K (p110_/_)/mTOR Pacliaxel Taxol ®,Onxes ™ C₄₇H₅₁NO₁₄ Inhibitors Vinorebine Vinorebine tartrate, C₄₅H₃₄N₄O₃Nevebine ® Rapamycin RAPA, Rapemune, C₃₁H₂₈NO₁₂ TGX-221 Siroimus, RPM,AY- C₂₁H₂₄N₄O₂ 22999 Histone Deacetylase & NU-7441 KU57788 C₂₅H₁₉NO₃SDNA-PK Inhibitors Trichostatin A TSA C₁₇H₂₂N₂O₃ STAT Inhibitors S31-201NSC 74859 C₁₈H₁₅NO₇S Antimetabolites Gemcitabine Gemzar ® C₃H₁₁F₂N₈O₄HClSurviving inhibitor LY2181308 Terameprecol YM155

Any of the disclosed compounds may be converted to a pharmaceuticallyacceptable salt. The pharmaceutically acceptable salt is notparticularly limited as long as it is non-toxic. Non-limiting examplesof salts with an inorganic or organic base include alkali metal salts(e.g. sodium salt, potassium salt and the like), alkaline earth metalsalts (e.g. calcium salt, magnesium salt and the like), ammonium salts,amine salts (e.g. triethylamine salt), and the like. Non-limitingexamples of acid addition salts derived from mineral acid (e.g.hydrochloride acid, hydrobromic acid, hydroiodic acid, phosphoric acid,nitric acid, sulphuric acid and the like), and salts derived fromorganic acids (e.g. tartaric acid, acetic acid, citric acid, malic acid,lactic acid, fumaric acid, maleic acid, benzoic acid, glycol acid,gluconic acid, succinic acid and the like).

Any of the disclosed compounds may be used as a prodrug for thebelow-mentioned pharmaceutical composition. As used herein, the term“prodrug” refers to any compound that can be converted to an active drugin vivo after administration, e.g. by being metabolized.

Administration of CIP2A dsRNAs and compounds of formula (I) may beconcomitant, simultaneous, or subsequent.

Delivery of CIP2A specific dsRNAs can be accomplished in two principallydifferent ways: 1) endogenous transcription of a nucleic acid sequenceencoding the oligonucleotide, where the nucleic acid sequence is locatedin an expression construct or 2) exogenous delivery of theoligonucleotide.

For endogenous transcription, CIP2A specific dsRNAs may be inserted intosuitable expression systems using methods known in the art. Non-limitingexamples of such expression systems include retroviral vectors,adenoviral vectors, lentiviral vectors, other viral vectors, expressioncassettes, and plasmids, such as those encapsulated in pegylatedimmunoliposomes (PILs), with or without one or more inducible promotersknown in the art. Both dsRNA strands may be expressed in a singleexpression construct from the same or separate promoters, or the strandsmay be expressed in separate expression constructs.

The above-mentioned expression systems may also be used for the deliveryof CIP2A silencing artificial miRNA precursors and shRNAs.

Typically, expression constructs are formulated into pharmaceuticalcompositions prior to administration to a human or animal subject (e.g.a canine subject). Administration may be performed by any suitablemethod known in the art, including systemic and local delivery. Theformulation depends on the intended route of administration as known toa person skilled in the art. By way of example, the expression constructmay be delivered in a pharmaceutically acceptable carrier or diluent, orit may be embedded in a suitable slow release composition. In somecases, the pharmaceutical composition may contain one or more cellsproducing the expression construct. Also bacteria may be used for RNAidelivery. For instance, recombinantly engineered Escherichia coli canenter mammalian cells after in vivo delivery and transfer shRNAs. Arelated approach is to use minicells derived e.g. from Salmonellaenterica.

For exogenous delivery, dsRNA molecules are typically complexed withliposome or lipid-based carriers, cholesterol conjugates, orpolyethyleneimine (PEI). A promising new approach is to complex dsRNAswith stable nucleic acid lipid particles (SNALPs). Suitable routes ofadministration for exogenous delivery, with or without said complexing,include, but are not limited to, parenteral delivery (e.g. intravenousinjection), enteral delivery (e.g. orally), local administration,topical administration (.e.g. dermally or transdermally) as known to aperson skilled in the art. Since surgical removal of a tumour is usuallythe primary clinical intervention, dsRNAs may be administered directlyto the resected tumour cavity.

Chemotherapeutic agents of formula (I) may be administered to a human oranimal subject by any suitable route known in the art including, but notlimited to, those listed for the administration of CIP2A specificdsRNAs.

In the present combination therapy, dsRNA molecules and compounds offormula (I) may be formulated into the same or separate pharmaceuticalcomposition. When separate pharmaceutical compositions are used,administration may be concomitant, simultaneous, or subsequent. Theformulation and/or route of administration for dsRNA molecules andcompounds of formula (I) may be selected independently from each other.In some embodiments, the pharmaceutical composition may comprise one ormore different CIP2A silencing dsRNAs and/or one or morechemotherapeutic agents of formula (I).

The pharmaceutical compositions may be administered in any appropriatepharmacological carrier suitable for administration. They can beadministered in any form that effect prophylactic, palliative,preventive or curing hyperproliferative diseases, such as cancer, inhuman or animal patients.

For the purposes of parenteral or topical administration, dsRNAs and/orcompounds of formula (I) may be formulated, for instance, as solutions,suspensions or emulsions. The formulations may comprise aqueous ornon-aqueous solvents, co-solvents, solubilizers, dispersing or wettingagents, suspending agents and/or viscosity agents, as needed.Non-limiting examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil, fish oil, and injectable organicesters. Aqueous carriers include, for instance, water, water-alcoholsolutions, including saline and buffered medial parenteral vehiclesincluding sodium chloride solution, Ringer's dextrose solution, dextroseplus sodium chloride solution, Ringer's solution containing lactose, orfixed oils. Non-limiting examples of intravenous vehicles include fluidand nutrient replenishers, electrolyte replenishers, such as those basedon Ringer's dextrose and the like. Aqueous compositions may comprisesuitable buffer agents, such as sodium and potassium phosphates,citrate, acetate, carbonate or glycine buffers depending on the targetedpH-range. The use of sodium chloride as a tonicity adjuster is alsouseful. The compositions may also include other excipients, such asstabilizing agents or preservatives. Useful stabilizing excipientsinclude surfactants (polysorbate 20 & 80, poloxamer 407), polymers(polyethylene glycols, povidones), carbohydrates (sucrose, mannitol,glucose, lactose), alcohols (sorbitol, glycerol propylene glycol,ethylene glycol), suitable proteins (albumin), suitable amino acids(glycine, glutamic acid), fatty acids (ethanolamine), antioxidants(ascorbic acid, cysteine etc.), chelating agents (EDTA salts, histidine,aspartic acid) or metal ions (Ca, Ni, Mg, Mn). Among useful preservativeagents are benzyl alcohol, chlorbutanol, benzalkonium chloride andpossibly parabens.

Solid dosage forms for oral administration include, but are not limitedto, capsules, tablets, pills, troches, lozenges, powders and granules.In such solid dosage forms, dsRNAs and/or compounds of formula (I) maybe admixed with at least one inert diluent such as sucrose, lactose orstarch. Such dosage forms may also comprise, as is normal practice,pharmaceutical adjuvant substances, e.g. stearate lubricating agents orflavouring agents. Solid oral preparations can also be prepared withenteric or other coatings which modulate release of the activeingredients.

Non-limiting examples of liquid dosage forms for oral administrationinclude pharmaceutically acceptable emulsions, solutions, suspensions,syrups and elixirs containing inert non-toxic diluents commonly used inthe art, such as water and alcohol. Such compositions may also compriseadjuvants, such as wetting agents, buffers, emulsifying, suspending,sweetening and flavouring agents.

The pharmaceutical composition may be provided in a concentrated form orin a form of a powder to be reconstituted on demand. In case oflyophilizing, certain cryoprotectants are preferred, including polymers(povidones, polyethylene glycol, dextran), sugars (sucrose, glucose,lactose), amino acids (glycine, arginine, glutamic acid) and albumin. Ifsolution for reconstitution is added to the packaging, it may consiste.g., of sterile water for injection or sodium chloride solution ordextrose or glucose solutions.

Means and methods for formulating the present pharmaceuticalpreparations are known to persons skilled in the art, and may bemanufactured in a manner which is in itself known, for example, by meansof conventional mixing, granulating, dissolving, lyophilizing or similarprocesses.

The present combination therapy may be used to treat human or animalhyperproliferative diseases including, but not limited to psoriasis,myocardial hypertrophy, benign tumors such as adenoma, hamartoma andchondroma, as well as cancers such as squamous cell carcinomas of thehead and neck, colon cancer, gastric cancer, breast cancer, ovariancancer, prostate cancer, cervical cancer, esophageal cancer, lungcancer, liver cancer, brain cancers (e.g. gliomas, astrocytomas, andglioblastomas), and haematological cancers (e.g. chronic and acuteT-cell and B-cell leukemias and lymphomas.).

As used herein, the term “treatment” or “treating” refers not only tocomplete cure of a disease, but also to prevention, alleviation, andamelioration of a disease or symptoms related thereto.

By an “efficient amount” of a combination of dsRNAs and compounds offormula (I) is meant an amount in which the harmful effects of a tumorare, at a minimum, ameliorated. Amounts and regimens for theadministration of the present combination therapy can be determinedreadily by those with ordinary skill in the clinical art of treatingcancer-related disorders. Generally, the dosage of the presentcombination therapy depend on considerations such as: age, gender andgeneral health of the patient to be treated; kind of concurrenttreatment, if any; frequency of treatment and nature of the effectdesired; extent of tissue damage; duration of the symptoms; and othervariables to be adjusted by the individual physician. A desired dose canbe administered in one or more applications to obtain the desiredresults. Pharmaceutical compositions according to the presentembodiments may be provided in unit dosage forms.

In one embodiment, dsRNAs may be administered in an effective amountwithin the dosage range of about 0.01 μg/kg to about 10 mg/kg, or about1.0 μg/kg to about 10 μg/kg. DsRNAs may be administered in a singledaily dose, or the total daily dosage may be administered in divideddoses, e.g. of two, three or four times daily. In one embodiment,compounds of formula (I) may be administered in an effective amountwithin the dosage range of about 0.1 μg/kg to about 300 mg/kg, or about1.0 μg/kg to about 10 mg/kg. The compounds of formula (I) may beadministered in a single daily dose, or the total daily dosage may beadministered in divided doses, e.g. of two, three or four times daily.The dosing schedule may be selected independently from the dosingschedule of dsRNAs.

It will be obvious to a person skilled in the art that, as thetechnology advances, the inventive concept can be implemented in variousways. The invention and its embodiments are not limited to the examplesdescribed below but may vary within the scope of the claims.

Example 1 CIP2A Inhibition Sensitizes T98G Cells to Various ChemotherapyDrugs

The human derived glioma cancer cells, T98G which express mutant p53,were transfected with either SCR siRNA (25 nM) or CIP2A siRNA (Seq ID#2; 25 nM). Following 48 h, media containing siRNA was replaced withmedia containing a chemotherapy drug at concentrations shown in FIG. 1.In order to determine if combining CIP2A inhibition with standardchemotherapy drugs would induce more potently apoptosis when compared tocells treated with either SCR siRNA or chemotherapy drugs alone, Caspase3/7 activity (Caspase 3/7 glo assay, Promega) which is used to determineapoptosis induction in cells, was measured 48 h later according to themanufacturer's instructions. The results shown in FIG. 1, demonstratethat CIP2A siRNA alone does not induce apoptosis. However, combiningCIP2A siRNA with either Lapatinib, Sunitinib; H-7; Vandetanib;Cisplatin; Paclitaxol; Temozolomide; Tandutinib or Indo)-3-carbinol,clearly enhanced the induction of apoptosis in T98G cells when comparedto cells treated with CIP2A siRNA alone.

Example 2 CIP2A Inhibition Sensitizes HeLa Cells to Various ChemotherapyDrugs

The human derived cervical cancer cells, HeLa in which wt p53 fuction isblunted by HPV18 E6, were transfected with either SCR siRNA (25 nM) orCIP2A siRNA (Seq ID #6; 25 nM). Following 72 h, media containing siRNAwas replaced with media containing a chemotherapy drug at concentrationsshown in FIG. 2. In order to determine if combining CIP2A inhibitionwith standard chemotherapy drugs would reduce cell viability morepotently when compared to cells treated with either SCR siRNA orchemotherapy drugs alone, the CTG assay (Promega) was used 48 h later,in accordance with the manufacturer's instructions. The results shown inFIG. 2, demonstrate that CIP2A siRNA alone has no effect on reducingcell viability. However, combining CIP2A siRNA with either Laptainib,PARPi (DPQ); Indo)-3-carbinol; NU-7441; Rapamycin; S31-201; TGX-221;Trichostatin A; Gemcitabine; or PKC-412 more potently reduced cellviabiltiy when compared to cells treated with CIP2A siRNA alone,indicating that inhibition of CIP2A sensitized HeLa cells to thesechemotherapeutic drugs.

Example 3 Primary Lymphoma Tumor Cells Derived from CIP2A Deficient(CIP2A^(−/−)) Mice are Sensitized to Chemotherapy Drugs

Primary mouse Lymphoma tumor cell lines which express mutant p53, werederived from the spleen of CIP2A wild type (CIP2A^(+/+)) or CIP2Adeficient (CIP2A^(−/−)) mice crossed with the Emu-myc mouse strain.Cells were seeded in 96-well plates and allowed to settle for 24 hoursbefore ‘normal growth’ media was replaced with media containingchemotherapy drug at concentrations shown in FIG. 3. In order todetermine if CIP2A deficient cancer cells were sensitized tochemotherapy drugs when compared with CIP2A expressing cancer cells,Caspase 3/7 activity (Caspase 3/7 glo assay, Promega) which is used todetermine apoptosis induction in cells was measured 48 h later accordingto the manufacturer's instructions. The results shown in FIG. 3,demonstrate that apoptosis is more potently induced in lymphoma cellsexpressing extremely low CIP2A levels (CIP2A−/−) when treated withvarious chemotherapy drugs including: Lapatinib; PARPi (DPQ); PKC-412;Tandutinib; Temozolomide; Paclitaxol; NU-7441; TGX-221 or S31-201 incomparison to CIP2A expressing cells.

Example 4 Primary Lymphoma Tumor Cells Derived from CIP2A Deficient(CIP2A−/−) Mice are Sensitized to Chemotherapy Drugs, Resulting inReduced Cell Viability

Primary mouse Lymphoma tumor cell lines which express mutant p53, werederived from the spleen of CIP2A wild type (CIP2A^(+/+)) or CIP2Adeficient (CIP2A^(−/−)) mice crossed with the Emu-myc mouse strain.Cells were seeded in 96-well plates and allowed to settle for 24 hoursbefore ‘normal growth’ media was replaced with media containingchemotherapy drug at concentrations as shown in FIG. 4. In order todetermine if treating CIP2A deficient cells with chemotherapy drugswould reduce cell viability when compared with CIP2A expressing cells,the CTG assay (Promega) was undertaken 48 h later according to themanufacturer's instructions. The results shown in FIG. 4, demonstratethat cell viability is more potently reduced in lymphoma cellsexpressing extremely low CIP2A levels (CIP2A−/−) treated with variouschemotherapy drugs including: Lapatinib; PARPi (DPQ); H-7; Tandutinib;PKC-312; Rapamycin; Trichostatin A; S31-201 or TGX-221 when compared tocells expressing CIP2A.

Example 5 CIP2A Inhibition Sensitizes MKN28 Cells to VariousChemotherapeutic Drugs

The human derived gastric cancer cells, MKN28, which express mutant p53were transfected with either SCR siRNA (25 nM) or CIP2A siRNA (Seq ID#6; 25 nM). Following 48 h, media containing siRNA was replaced withmedia containing a chemotherapy drug at concentrations shown in FIG. 5.In order to determine if combining CIP2A inhibition with standardchemotherapy drugs would induce apoptosis more potently when compared tocells treated with either SCR siRNA or chemotherapy drugs alone, Caspase3/7 activity (Caspase 3/7 glo assay, Promega) which is used to determineapoptosis induction in cells, was measured 48 h later according to themanufacturer's instructions. The results shown in FIG. 5, demonstratethat CIP2A siRNA alone induced apoptosis when compared to MKN28 cellstreated with SCR siRNA alone. However, the level of apoptosis is clearlyenhanced when combining CIP2A siRNA with various chemotherapy drugsincluding: H-7, Vandetanib, Cisplatin, Paclitaxol, Temozolomide,Gemcitabine, PKC-412, Indol-3-carbinol and Tandutinib.

Example 6 CIP2A Inhibition does not Sensitize MCF-7 Cells, which ExpressWild Type p53, to Chemotherapeutic Drugs

The human derived breast cancer cells, MCF-7, which express wild typep53 were transfected with either SCR siRNA (25 nM) or CIP2A siRNA (SeqID #2; 25 nM). Following 48 h, media containing siRNA was replaced withmedia containing a chemotherapy drug at concentrations shown in FIG. 6.In order to determine if combining CIP2A inhibition with standardchemotherapy drugs would induce apoptosis significantly when compared tocells treated with either SCR siRNA or chemotherapy drugs alone, Caspase3/7 activity (Caspase 3/7 glo assay, Promega) which is used to determineapoptosis induction in cells, was measured 48 h later according to themanufacturer's instructions. The results shown in FIG. 6, demonstratethat CIP2A siRNA alone did not induce apoptosis when compared to MCF-7cells treated with SCR siRNA alone. Similarly, combining CIP2A siRNAwith various chemotherapeutic drugs did not induce apoptosis in thesecells expressing wild-type p53.

REFERENCES

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1.-15. (canceled)
 16. A pharmaceutical composition comprising acombination of at least one type of a CIP2A silencing agent selectedfrom the group consisting of an siRNA molecule, DsiRNA molecule,artificial miRNA precursor, shRNA molecule, antisense oligonucleotide,and ribozyme; and a compound selected from the group consisting ofPKC-412, PARP inhibitor III, indol-3-carbinol, cisplatin, rapamycin,TGX-221, NU-7441, S31-201, and gemcitabine; and at least onepharmaceutically acceptable carrier.
 17. The pharmaceutical compositionaccording to claim 16, wherein the CIP2A silencing agent comprises anucleic acid sequence selected from the group consisting of SEQ ID NO:s1 to 25, and sequences having at least 80% sequence identity to said SEQID NO:s 1 to 25 and retaining their CIP2A silencing activity.
 18. Thepharmaceutical composition according to claim 16, for use in thetreatment of a hyperproliferative disease selected from a groupconsisting of psoriasis, myocardial hypertrophy, benign tumors, solidcancers and haematological cancers.
 19. The pharmaceutical compositionaccording to claim 18, wherein said solid cancer is selected from agroup consisting of squamous cell carcinomas of the head and neck, coloncancer, gastric cancer, breast cancer, ovarian cancer, prostate cancer,cervical cancer, esophageal cancer, lung cancer, liver cancer, braincancer, glioma, astrocytoma, and glioblastoma and haematological cancersis selected from a group consisting of acute and chronic T-cell andB-cell leukemias and lymphomas.
 20. A method of sensitizinghyperproliferative cells with impaired p53 function to achemotherapeutic agent by silencing CIP2A gene in a human or animalsubject in need of such sensitization by administering at least one typeof CIP2A silencing agent selected from the group consisting of an siRNAmolecule, DsiRNA molecule, artificial miRNA precursor, shRNA molecule,antisense oligonucleotide, and ribozyme, and a compound selected fromthe group consisting of PKC-412, PARP inhibitor III, indol-3-carbinol,cisplatin, rapamycin, TGX-221, NU-7441, S31-201, and gemcitabine.
 21. Amethod of treating a hyperproliferative disease comprising cells withimpaired p53 function in a human or animal subject in need of suchtreatment by administering at least one type of CIP2A silencing agentselected from the group consisting of an siRNA molecule, DsiRNAmolecule, artificial miRNA precursor, shRNA molecule, antisenseoligonucleotide, and ribozyme; and a compound selected from the groupconsisting of: PKC-412, PARP inhibitor III, indol-3-carbinol, cisplatin,rapamycin, TGX-221, NU-7441, S31-201, and gemcitabine, concomitantly,simultaneously, or subsequently to said subject.
 22. The methodaccording to claim 21, wherein the CIP2A silencing agent comprises anucleic acid sequence selected from the group consisting of SEQ ID NO:s1 to 25 and sequences having at least 80% sequence identity to said SEQID NO:s 1 to 25 and retaining their CIP2A silencing activity.
 23. Themethod according to claim 21, for use in the treatment of ahyperproliferative disease selected from a group consisting ofpsoriasis, myocardial hypertrophy, benign tumors, solid cancers andhaematological cancers.
 24. The method according to claim 23, whereinsaid solid cancer is selected from a group consisting of squamous cellcarcinomas of the head and neck, colon cancer, gastric cancer, breastcancer, ovarian cancer, prostate cancer, cervical cancer, brain cancer,glioma, astrocytoma, and glioblastoma.
 25. A method of selecting acancer therapy for a subject in need of such therapy, wherein the methodcomprises evaluating CIP2A and p53 expression and/or protein activity ina sample obtained from said subject, and selecting monotherapy by atleast one chemotherapeutic agent for subjects whose sample is negativefor CIP2A expression and/or activity and impaired for p53 activity, andselecting the combination according to claim 16 as a therapy forsubjects whose sample is positive for CIP2A expression and impaired forp53 activity.
 26. The method according to claim 25, wherein said subjectsuffers from a hyperproliferative disease selected from a groupconsisting of psoriasis, myocardial hypertrophy, benign tumors, solidcancers and haematological cancers.
 27. A kit comprising reagents forthe practice of the method according to claim 25.